Molecular and physiological analysis of Anopheles funestus swarms in Nchelenge, Zambia.

BACKGROUND: Anopheles funestus has been recognized as a major malaria vector in Africa for over 100 years, but knowledge on many aspects of the biology of this species is still lacking. Anopheles funestus, as with most other anophelines, mate through swarming. A key event that is crucial for the An. funestus male to mate is genitalia rotation. This involves the 135° to 180° rotation of claspers, which are tipped with claws. This physical change then enables the male to grasp the female during copulation. The aim of this investigation was to molecularly characterize wild An. funestus swarms from Zambia and examine the degree of genitalia rotation within the swarm. METHODS: Anopheles funestus swarms were collected from Nchelenge, northern Zambia, during dusk periods in May 2016. All the adults from the swarm were analysed morphologically and identified to species level using a multiplex PCR assay. Anopheles funestus s.s. specimens were molecularly characterized by restriction fragment length polymorphism type and Clade type assays. The different stages of genitalia rotation were examined in the adult males. RESULTS: A total of six swarms were observed during the study period and between 6 and 26 mosquitoes were caught from each swarm. Species analysis revealed that 90% of the males from the swarms were An. funestus s.s. MW-type, with 84% belonging to clade I compared to 14% clade II and 2% failed to amplify. Very few specimens (3.4%) were identified as Anopheles gambiae s.s. Eighty percent of the males from the swarm had complete genitalia rotation. CONCLUSIONS: This is the first time that An. funestus swarms have been molecularly identified to species level. Anopheles funestus swarms appear to be species-specific with no evidence of clade-type differentiation within these swarms. The An. funestus swarms consist mainly of males with fully rotated genitalia, which strongly suggests that swarming behaviour is triggered primarily when males have matured.

Does mosquito mass-rearing produce an inferior mosquito?

BACKGROUND: The success of the sterile insect technique depends, among other things, on continuous releases of sexually competitive sterile males within the target area. Several factors (including high rearing density and physical manipulation, such as larvae and pupae separation) can influence the quality of males produced in mass-rearing facilities. The different steps in mass production in the laboratory may modify the behaviour of mosquitoes, directly or through loss of natural characters as a result of adaptation to lab rearing, and lead to the competitiveness of sterile male being reduced. In the present study, the objective was to evaluate the effect of mass-rearing conditions on sterile male sexual competitiveness in semi-field cages compared to routine small scale laboratory rearing methods. METHODS: Anopheles arabiensis immature stages were reared both on a large scale using a rack and tray system developed by the FAO/IAEA (MRS), and on a small scale using standard laboratory rearing trays (SRS). Mosquito life history traits such as pupation rate, emergence rate, adult size as well as the effect of irradiation on adult longevity were evaluated. Moreover, 5-6 day old mosquitoes were released into field cages and left for two nights to mate and the mating competitiveness between sterile mass-reared males and fertile males reared on a small scale when competing for small scale reared virgin females was investigated. Resulting fertility in a treatment ratio of 1:1:1 (100 irradiated males: 100 non-irradiated males: 100 virgin females) was compared to control cages with 0:100:100 (non-irradiated control) and 100:0:100 (irradiated control). RESULTS: No significant differences in life history parameters were observed between rearing methods. The competitiveness index of mass reared males (0.58) was similar to males reared on a small scale (0.59). A residual fertility rate of 20% was observed in the irradiated control (100:0:100), measured as the percentage of eggs collected from the cages which developed to adulthood. No significant difference was observed (t = 0.2896, df = 4, P = 0.7865) between the rearing treatments (MRS and SRS) in the fertility rate, a measure of mating competitiveness. CONCLUSIONS: The results showed that the FAO/IAEA mass-rearing process did not affect mosquito life history parameters or the mating competitiveness of males.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors.

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

The anticancer human mTOR inhibitor sapanisertib potently inhibits multiple Plasmodium kinases and life cycle stages.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

New distribution record of Anopheles rivulorum-like from Sadiola, Mali, with notes on malaria vector insecticide resistance.

BACKGROUND: The SEMOS gold mine in Sadiola, southwestern Mali, has been implementing a malaria vector control programme for 15 y using indoor residual house spraying and sporadic larval control. Periodic screening of the vector populations have been carried out over the years to provide information to the control programme, mainly on vector species present and their insecticide resistance status. The data from five entomological surveys, carried out in 2006, 2011, 2014, 2016 and 2018, are presented. METHODS: Adult mosquitoes were collected resting on walls inside houses and on verandas. Insecticide susceptibility assays were carried out and mosquitoes subsequently identified by species using molecular assays. RESULTS: The major malaria vector mosquitoes, Anopheles gambiae and Anopheles arabiensis were abundant at each sampling period with Anopheles coluzzii and Anopheles funestus being rare or absent. Anopheles rivulorum was identified in 2006 and Anopheles leesoni in 2016. The presence of Anopheles rivulorum-like, identified for the first time in 2018, was not screened for in previous surveys. Insecticide susceptibility bioassays showed resistance in both A. gambiae and A. arabiensis to pyrethroids, carbamates and dichlorodiphenyltrichloroethane over the 12 y. CONCLUSIONS: This is the first record of A. rivulorum-like west of Côte d'Ivoire. Resistance levels to the three classes of insecticides were variable but appeared to decrease after pyrethroids were discontinued for house spraying.

Benzimidazole Derivatives Are Potent against Multiple Life Cycle Stages of Plasmodium falciparum Malaria Parasites.

The continued emergence of resistance to front-line antimalarial treatments is of great concern. Therefore, new compounds that potentially have a novel target in various developmental stages of Plasmodium parasites are needed to treat patients and halt the spread of malaria. Here, several benzimidazole derivatives were screened for activity against the symptom-causing intraerythrocytic asexual blood stages and the transmissible gametocyte stages of P. falciparum. Submicromolar activity was obtained for 54 compounds against asexual blood stage parasites with 6 potent at IC50 < 100 nM while not displaying any marked toxicity against mammalian cells. Nanomolar potency was also observed against gametocytes with two compounds active against early stage gametocytes and two compounds active against late-stage gametocytes. The transmission-blocking potential of the latter was confirmed as they could prevent male gamete exflagellation and the lead compound reduced transmission by 72% in an in vivo mosquito feeding model. These compounds therefore have activity against multiple stages of Plasmodium parasites with potential for differential targets.

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

Identification and Profiling of a Novel Diazaspiro[3.4]octane Chemical Series Active against Multiple Stages of the Human Malaria Parasite Plasmodium falciparum and Optimization Efforts.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

A standardised method of marking male mosquitoes with fluorescent dust.

BACKGROUND: Prior to a major release campaign of sterile insects, including the sterile insect technique, male mosquitoes must be marked and released (small scale) to determine key parameters including wild population abundance, dispersal and survival. Marking insects has been routinely carried out for over 100 years; however, there is no gold standard regarding the marking of specific disease-transmitting mosquitoes including Anopheles arabiensis, Aedes aegypti and Aedes albopictus. The research presented offers a novel dusting technique and optimal dust colour and quantities, suitable for small-scale releases, such as mark-release-recapture studies. METHODS: We sought to establish a suitable dust colour and quantity for batches of 100 male An. arabiensis, that was visible both by eye and under UV light, long-lasting and did not negatively impact longevity. A set of lower dust weights were selected to conduct longevity experiments with both Ae. aegypti and Ae. albopictus to underpin the optimal dust weight. A further study assessed the potential of marked male An. arabiensis to transfer their mark to undusted males and females. RESULTS: The longevity of male An. arabiensis marked with various dust colours was not significantly reduced when compared to unmarked controls. Furthermore, the chosen dust quantity (5 mg) did not negatively impact longevity (P = 0.717) and provided a long-lasting mark. Dust transfer was found to occur from marked An. arabiensis males to unmarked males and females when left in close proximity. However, this was only noticeable when examining individuals under a stereomicroscope and thus deemed negligible. Overall, male Ae. aegypti and Ae. albopictus displayed a greater sensitivity to dusting. Only the lowest dust weight (0.5 mg) did not significantly reduce longevity (P = 0.888) in Ae. aegypti, whilst the lowest two dust weights (0.5 and 0.75 mg) had no significant impact on longevity (P = 0.951 and 0.166, respectively) in Ae. albopictus. CONCLUSION: We have devised a fast, inexpensive and simple marking method and provided recommended dust quantities for several major species of disease-causing mosquitoes. The novel technique provides an evenly distributed, long-lasting mark which is non-detrimental. Our results will be useful for future MRR studies, prior to a major release campaign.

Anopheline Mosquito Species Composition, Kdr Mutation Frequency, and Parasite Infectivity Status in Northern Tanzania.

The scaling-up of malaria control interventions in northern Tanzania has resulted in a decline in malaria prevalence and vector species composition. Despite this achievement, residual malaria transmission remains a concern in the area. The main aim of this study was to investigate malaria vector species composition, parasite infectivity rates, and the presence of insecticide knockdown resistance (kdr) mutations in three sites that have experienced a significant decline in malaria in northern Tanzania. Adult mosquitoes were sampled using light traps in houses and hand-aspirators in cowsheds, whereas the standard dipping method was used for sampling mosquito larvae. Adult mosquitoes identified as Anopheles gambiae s.l. and An. funestus s.l. and larval stages III and IV of An. gambiae s.l. were stored in absolute ethanol for further laboratory molecular identification. The identified species in the An. gambiae complex were An. gambiae s.s., An. merus, An. quadriannulatus, and An. arabiensis, whereas the An. funestus group comprised An. funestus s.s., An. rivulorum, and An. leesoni. For An. gambiae s.s. analyzed from Zeneth, 47.6% were kdr-East homozygous susceptible, 35.7% kdr-East heterozygous resistant, 9.6% kdr-East homozygous resistant, and 7.1% undefined, whereas specimens from Kwakibuyu were 45.5% kdr-East homozygous susceptible, 32.7% kdr-East heterozygous resistant, 16.3% kdr-East homozygous resistant, and 5.5% undefined. There were no kdr-West alleles identified from any specimen. The overall malaria parasite infectivity rate was 0.75%. No infections were found in Moshi. The findings indicate that populations of the major malaria vector mosquitoes are still present in the study area, with An. funestus taking a lead in malaria transmission.

Benchmarking insecticide resistance intensity bioassays for Anopheles malaria vector species against resistance phenotypes of known epidemiological significance.

BACKGROUND: Insecticide use via indoor residual spraying (IRS) or treated nets is the primary method for controlling malaria vector populations. The incidence of insecticide resistance in vector populations is burgeoning globally making resistance management key to the design of effective malaria control and elimination strategies. Vector populations can be assessed for insecticide resistance using a binary (susceptible or resistant) classification based on the use of the standard WHO insecticide susceptibility assay for adult anopheline mosquitoes. However, the recent scaling up of vector control activities has necessitated a revision of the WHO bioassay protocol to include the production of information that not only diagnoses resistance but also gives information on the intensity of expression of resistance phenotypes detected. This revised protocol is expected to inform on the range of resistance phenotypes in a target vector population using discriminating/diagnostic insecticide concentrations (DC) as well as their potential operational significance using 5× DC and 10× DC assays. The aim of this project was to use the revised protocol to assess the intensity of pyrethroid resistance in a range of insecticide resistant Anopheles strains with known resistance mechanisms and for which there is evidence of operational significance in the field setting from which these colonies were derived. METHODS: Diagnostic concentration (DC) bioassays followed by 5× DC and 10× DC assays using the pyrethroid insecticides permethrin and deltamethrin were conducted according to the standard WHO bioassay method against pyrethroid resistant laboratory strains of Anopheles funestus, An. arabiensis and An. gambiae. RESULTS: Low to moderate resistance intensities were recorded for the An. arabiensis and An. gambiae strains while moderate to high intensities were recorded for the An. funestus strains. CONCLUSIONS: It is evident that resistance intensity assays can add predictive value to the decision making process in vector control settings, although more so in an IRS setting and especially when bench-marked against resistance phenotypes of known operational significance.